Fibrinogen is a precursor of fibrin, which is a predominant protein that forms blood clots, and is produced in liver parenchyma cells. In the human body, about 80% of fibrinogen is present in plasma (e.g., about 200 to 400 mg/dL in a healthy adult), and the remaining amount is present in tissues. Fibrinogen is a glycoprotein containing three paired polypeptide (i.e., Aα, Bβ and γ) chains coupled via a disulfide bond. When the Aα chains and the Bβ chains are cleaved from fibrinogen by thrombin, fibrin is formed. Fibrin plays the main role in thrombus formation and hemostasis. Clinically, fibrinogen increases under the condition of inflammation, and decreases in severe hepatic disorders, DIC, etc.
Fibrinogen can be assayed through the Clauss method (thrombin addition method), the PT-derived method (clotting time method), TIA (tubidimetric immunoassay), the latex method, etc. Among them, the thrombin addition method is commonly employed. Specifically, thrombin and calcium chloride are added to a sodium citrate-added plasma sample, and the clotting time is measured. The fibrinogen concentration (mg/dL) in the sample is calculated by a calibration curve obtained from clotting times at known fibrinogen concentrations.
A thrombin employed in fibrinogen assay is α-thrombin, which is produced through cleavage of a peptide from prothrombin (i.e., precursor of thrombin) and exhibits a clotting activity. The α-thrombin is known to undergo self-decomposition to form β-thrombin, or further to form γ-thrombin, which are low molecular weight thrombins with no clotting activity. Therefore, α-thrombin is generally lyophilized for long-term storage.
Several attempts have been made to stabilize thrombin in a solution. For example, there have been known several methods such as: (1) a method in which high-concentration glycerol and polyol (e.g., sucrose, mannitol, sorbitol, etc.) are added to thrombin (Patent Document 1); and (2) a method in which saccharide, amino acid, etc. are added to thrombin (Patent Document 2).
However, these methods exhibit an unsatisfactory effect of stabilizing thrombin in a liquid fibrinogen assay reagent.
One known stabilization method for thrombin contained in a fibrinogen assay reagent includes adding a thrombin antagonist to a thrombin-containing solution (Patent Document 3).
However, the mechanism of this method is based on inhibition of the activity of thrombin. Therefore, when this method is employed, the clotting time of a low-fibrinogen-concentration sample, which is an important sample in fibrinogen assay, is considerably prolonged, and in some cases not detectable. Prolongation of the clotting time causes reduction in sample throughput and failure in assaying of low-fibrinogen-concentration samples causes an increase in the number of samples to be re-assayed. Thus, the above method is not suited for an emergency medical test.
Under such circumstances, there is demand for a method for stabilizing α-thrombin in an α-thrombin-containing solution which can be performed on low-fibrinogen-concentration samples without causing considerable prolongation of assay time and with little effects on measurements, thus being applicable to liquid fibrinogen assay reagents.
Patent Document 1: JP-A-1987-106028
Patent Document 2: JP-A-1989-040433
Patent Document 3: JP-A-2004-191367